Pancreatic cancers, about 95% of which are pancreatic ductal adenocarcinoma (PDAC), have the worst prognosis of all cancers and will soon be the second leading cause of cancer-related deaths in the United States. Current treatment regimens clearly fail to benefit patient survival. Oncolytic virotherapy is a relatively new anticancer approach that utilizes replication-competent viruses to specifically infect and kill tumor cells. Vesicular stomatitis virus (VSV) is one such oncolytic virus (OV) and is already in a phase I clinical trial against hepatocellular carcinoma. Our recent studies demonstrated that VSV is effective against the majority of tested human PDAC cell lines, both in vitro and in vivo. However, we found that some PDAC cell lines are resistant to VSV infection, replication and virus-mediated oncolysis, largely due to the constitutive expression of antiviral genes. Our goal is to better understand and target PDAC cellular pathways determining the success of VSV- based OV therapy and to develop rational combination therapy approaches to enhance OV efficacy and/or overcome resistance to OVs. The proposed experiments are based on our recently published work and preliminary studies that analyzed VSV against a number of PDAC cell lines in vitro and in vivo, made an initial characterization of PDAC cells lines in regard to their general cellular characteristics and permissiveness to VSV, and identified small molecule inhibitors dramatically improving VSV performance in PDAC cells. In Aim 1, we will identify and target cellular factors leading to the constitutive expression of antiviral genes in PDAC cell line resistant to VSV; identify specific antiviral genes responsible for resistance of PDAC cell lines t VSV; and investigate how development of chemoresistance affects permissiveness of PDAC cells to VSV. In Aim 2, we will test in vivo VSV in combination with small molecule inhibitors shown in our preliminary studies to successfully down-regulate antiviral genes and stimulate VSV replication and oncolysis. Importantly, both aims will evaluate how these novel combination approaches affect oncoselectivity and safety of VSV-based OV therapy. Our studies will develop a more effective VSV oncolytic therapy, broaden the spectrum of PDAC phenotypes to which this treatment can be successfully applied, and will lead to development of prescreening approaches to identify cancer patients who will be responsive to specific OV treatments. While this proposal is focused on VSV against PDAC, it will define cellular factors and pathways likely affecting other OVs and tumor systems.